Chondrogenic differentiation of placenta-derived mesenchymal stem cells in vitro

doi:10.3969/j.issn.2095-4344.2014.32.020

Abstract

Abstract:

BACKGROUND: Placenta-derived mesenchymal stem cells have been shown to have a strong proliferative capacity, and can differentiate into osteoblasts, neuroblasts, hepatocyte-like cells, but there are few studies about their chondrogenic differentiation.
OBJECTIVE: To induce the differentiation of human placenta-derived mesenchymal stem cells into chondrocytes in vitro.
METHODS: Human placenta-derived mesenchymal stem cells were culture in vitro and identified. Passage 3 cells were used to prepare cell suspensions at the concentration of 1.6×1010 cells/L, and then cultured in 24-well plates at a volume of 5, 10, 15 μL for 2 hours for the purpose of forming micelles. After that, the samples were cultured in mesenchymal stem cell medium or cartilage induction medium for 14 days. Alcian blue staining, general observation and inverted microscope observation were performed.
RESULTS AND CONCLUSION: A great amount of adherent cells formed in the mesenchymal stem cell medium, which possessed typical morphology of mesenchymal stem cells. After cultured in cartilage induction medium, the cells only maintained micelles and did not proliferate continuously to form adherent cells. The base of these micelles had no adherent cells, and the diameter of micelles became increasing with the increasing number of inoculated cells. These findings suggest that human placenta-derived mesenchymal stem cells have the chondrogenic ability, and can be used as seed cells for tissue engineering and cell therapy.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: stem cells, embryonic stem cells, placenta, chondrocytes

CLC Number:

R394.2

Li Zhi, Zhao Wei, Liu Wei, Zhou Ye, Jia Jing-qiao, Yang Li-feng . Chondrogenic differentiation of placenta-derived mesenchymal stem cells in vitro[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(32): 5203-5208.

Figures/Tables 1

2.1 胎盘来源间充质干细胞形态及细胞表面标志物检测结果人胎盘间充质干细胞在间充质干细胞培养基中培养，细胞增殖形成大量贴壁细胞，放大40倍倒置显微镜观察，贴壁细胞具有典型的间充质细胞形态，即纺锤体型，折光性强。用流式细胞仪检测结果显示，阳性表达CD90、CD44、CD105和CD73，不表达CD34、CD14和CD45。流式细胞仪检测结果表明实验培养的细胞具备干细胞的表面标志物，可证实为胎盘间充质干细胞。 2.2 成软骨细胞诱导后阿利新蓝染色结果倒置显微镜观察：对照组1：形成微团时所用的胎盘来源间充质干细胞的细胞悬液体积为5 μL，细胞悬液浓度为1.6×1010 L-1，在间充质干细胞培养基中培养，每3 d换液1次。细胞增殖形成大量贴壁细胞，14 d后阿利新蓝染色，放大40倍倒置显微镜观察，贴壁细胞具有典型的间充质细胞形态，即纺锤体型，折光性强，色成淡蓝色(图1A)。对照组2：形成微团时用细胞浓度为1.6×1010 L-1的胎盘来源间充质干细胞悬液10 μL，可见细胞增殖形成大量贴壁细胞外，还会形成细胞膜状反折，14 d后阿利新蓝染色，倒置显微镜观察，贴壁细胞具有典型的间充质细胞形态(图1B)。对照组3：形成微团时用细胞浓度为1.6×1010 L-1的胎盘来源间充质干细胞悬液15 μL，4 d后行阿利新蓝染色，倒置显微镜观察，微团的基部具有大量贴壁细胞，微团不圆润(图1C)。实验组1：形成微团时所用的胎盘来源间充质干细胞的细胞悬液体积为5 μL，细胞悬液浓度为1.6×1010 L-1，在软骨诱导培养基中培养，每3 d换液1次。14 d后行阿利新蓝染色，放大40倍倒置显微镜观察，细胞只会保持微团，不会继续增殖形成贴壁细胞，微团基部无贴壁细胞，微团悬浮培养基中，微团直径约1 mm。倒置显微镜下无法观察到微团内的细胞形态，但可观察到实验组中微团的边缘向外延伸出少数的丝状细胞结构(图1D)。实验组2，3：形成微团时用细胞浓度为1.6×1010 L-1的胎盘来源间充质干细胞悬液10，15 μL，倒置显微镜观察悬浮培养基中的微团直径约1.5，2 mm，其形态与实验组1相似(图1E，F)。大体观察结果：各组胎盘来源间充质干细胞的数码相机照相结果见图2。对照组1，2中细胞在间充质干细胞培养基中增殖良好，形成大量贴壁细胞长满24孔板(图2A，B)；对照组3中有细胞团出现(图2C)；实验组细胞在软骨诱导培养基中培养，未见贴壁细胞，微团直径约1.0，1.5，2.0 mm(图2D-F)。 "

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